Compounds and methods for the treatment of cancer

ABSTRACT

(−)-(2S,4S)-1-(2-Hydroxymethyl-1,3-dioxolan-4-yl)cytosine (also referred to as (−)-OddC) or its derivative and its use to treat cancer in animals, including humans.

This application is a continuation-in-part of U.S. Ser. No. 08/301,298,entitled Compounds and Methods for the Treatment of Cancer, filed onSep. 6, 1994, by Yung-Chi Cheng, Chung K. Chu which is acontinuation-in-part of U.S. application Ser. No. 07/937,845, filed onOct. 19, 1992, by Yung-Chi Cheng, Chung K. Chu, Hea O. Kim, andKirupathevy Shanmuganathan, which is entitled “Method of Treating orPreventing Hepatitis B Virus” which claims priority to PCT/US92/03144,filed on Apr. 16, 1992.

GOVERNMENT RIGHTS

The U.S. government has rights in this invention by virtue of Grant No.CA-44358 from the National Cancer Institute (NIH).

FIELD OF THE INVENTION

This invention is in the area of medicinal chemistry, and in particularis (−)-(2S,4S)-1-(2-hydroxymethyl-1,3-dioxolan-4-yl)cytosine (alsoreferred to as (−)-OddC) or its derivative, and its use to treat cancerin animals, including humans.

BACKGROUND OF THE INVENTION

A tumor is an unregulated, disorganized proliferation of cell growth. Atumor is malignant, or cancerous, if it has the properties ofinvasiveness and metastasis. Invasiveness refers to the tendency of atumor to enter surrounding tissue, breaking through the basal laminasthat define the boundaries of the tissues, thereby often entering thebody's circulatory system. Metastasis refers to the tendency of a tumorto migrate to other areas of the body and establish areas ofproliferation away from the site of initial appearance.

Cancer is now the second leading cause of death in the United States.Over 8,000,000 persons in the United States have been diagnosed withcancer, with 1,208,000 new diagnoses expected in 1994. Over 500,000people die annually from the disease in this country.

Cancer is not fully understood on the molecular level. It is known thatexposure of a cell to a carcinogen such as certain viruses, certainchemicals, or radiation, leads to DNA alteration that inactivates a“suppressive” gene or activates an “oncogene”. Suppressive genes are.growth regulatory genes, which upon mutation, can no longer control cellgrowth. Oncogenes are initially normal genes (called prooncogenes) thatby mutation or altered context of expression become transforming genes.The products of transforming genes cause inappropriate cell growth. Morethan twenty different normal cellular genes can become oncogenes bygenetic alteration. Transformed cells differ from normal cells in manyways, including cell morphology, cell-to-cell interactions, membranecontent, cytoskeletal structure, protein secretion, gene expression andmortality (transformed cells can grow indefinitely).

All of the various cell types of the body can be transformed into benignor malignant tumor cells. The most frequent tumor site is lung, followedby colorectal, breast, prostate, bladder, pancreas, and then ovary.Other prevalent types of cancer include leukemia, central nervous systemcancers, including brain cancer, melanoma, lymphoma, erythroleukemia,uterine cancer, and head and neck cancer.

Cancer is now primarily treated with one or a combination of three typesof therapies: surgery, radiation, and chemotherapy. Surgery involves thebulk removal of diseased tissue. While surgery is sometimes effective inremoving tumors located at certain sites, for example, in the breast,colon, and skin, it cannot be used in the treatment of tumors located inother areas, such as the backbone, nor in the treatment of disseminatedneoplastic conditions such as leukemia.

Chemotherapy involves the disruption of cell replication or cellmetabolism. It is used most often in the treatment of leukemia, as wellas breast, lung, and testicular cancer.

There are five major classes of chemotherapeutic agents currently in usefor the treatment of cancer: natural products and their derivatives;anthracyclines; alkylating agents; antiproliferatives (also calledantimetabolites); and hormonal agents. Chemotherapeutic agents are oftenreferred to as antineoplastic agents.

The alkylating agents are believed to act by alkylating andcross-linking guanine and possibly other bases in DNA, arresting celldivision. Typical alkylating agents include nitrogen mustards,ethyleneimine compounds, alkyl sulfates, cisplatin, and variousnitrosoureas. A disadvantage with these compounds is that they not onlyattack malignant cells, but also other cells which are naturallydividing, such as those of bone marrow, skin, gastrointestinal mucosa,and fetal tissue.

Antimetabolites are typically reversible or irreversible enzymeinhibitors, or compounds that otherwise interfere with the replication,translation or transcription of nucleic acids.

Several synthetic nucleosides have been identified that exhibitanticancer activity. A well known nucleoside derivative with stronganticancer activity is 5-fluorouracil. 5-Fluorouracil has been usedclinically in the treatment of malignant tumors, including, for example,carcinomas, sarcomas, skin cancer, cancer of the digestive organs, andbreast cancer. 5-Fluorouracil, however, causes serious adverse reactionssuch as nausea, alopecia, diarrhea, stomatitis, leukocyticthrombocytopenia, anorexia, pigmentation, and edema. Derivatives of5-fluorouracil with anti-cancer activity have been described in U.S.Pat. No. 4,336,381, and in Japanese patent publication Nos. 50-50383,50-50384, 50-64281, 51-146482, and 53-84981.

U.S. Pat. No. 4,000,137 discloses that the peroxidate oxidation productof inosine, adenosine, or cytidine with methanol or ethanol has activityagainst lymphocytic leukemia.

Cytosine arabinoside (also referred to as Cytarabin, araC, and Cytosar)is a nucleoside analog of deoxycytidine that was first synthesized in1950 and introduced into clinical medicine in 1963. It is currently animportant drug in the treatment of acute myeloid leukemia. It is alsoactive against acute lymphocytic leukemia, and to a lesser extent, isuseful in chronic myelocytic leukemia and non-Hodgkin's lymphoma. Theprimary action of araC is inhibition of nuclear DNA synthesis.Handschumacher, R. and Cheng, Y., “Purine and PyrimidineAntimetabolites”, Cancer Medicine, Chapter XV-1, 3rd Edition, Edited byJ. Holland, et al., Lea and Febigol, publishers.

5-Azacytidine is a cytidine analog that is primarily used in thetreatment of acute myelocytic leukemia and myelodysplastic syndrome.

2-Fluoroadenosine-5′-phosphate (Fludara, also referred to as FaraA)) isone of the most active agents in the treatment of chronic lymphocyticleukemia. The compound acts by inhibiting DNA synthesis. Treatment ofcells with F-araA is associated with the accumulation of cells at theG1/S phase boundary and in S phase; thus, it is a cell cycle Sphase-specific drug. Incorporation of the active metabolite, F-araATP,retards DNA chain elongation. F-araA is also a potent inhibitor ofribonucleotide reductase, the key enzyme responsible for the formationof DATP.

2-Chlorodeoxyadenosine is useful in the treatment of low grade B-cellneoplasms such as chronic lymphocytic leukemia, non-Hodgkins' lymphoma,and hairy-cell leukemia. The spectrum of activity is similar to that ofFludara. The compound inhibits DNA synthesis in growing cells andinhibits DNA repair in resting cells.

Although a number of chemotherapeutic agents have been identified andare currently used for the treatment of cancer, new agents are soughtthat are efficacious and which exhibit low toxicity toward healthycells.

Therefore, it is an object of the present invention to provide compoundsthat exhibit anti-tumor, and in particular, anti-cancer, activity.

It is another object of the present invention to provide pharmaceuticalcompositions for the treatment of cancer.

It is further object of the present invention to provide a method forthe treatment of cancer.

SUMMARY OF THE INVENTION

A method and composition for the treatment of cancer in humans and otherhost animals is disclosed that includes administering an effectiveamount of (−)-(2S,4S)-1-(2-hydroxymethyl-1,3-dioxolan-4-yl)cytosine(also referred to as (−)-OddC), a pharmaceutically acceptable derivativethereof, including a 5′ or N⁴ alkylated or acylated derivative, or apharmaceutically acceptable salt thereof, optionally in apharmaceutically acceptable carrier.

In a preferred embodiment,(−)-(2S,4S)-1-(2-hydroxymethyl-1,3-dioxolan-4-yl)cytosine is provided asthe indicated enantiomer and substantially in the absence of itscorresponding enantiomer (i.e., in enantiomerically enriched, includingenantiomerically pure form).

It is believed that(−)-(2S,4S)-1-(2-hydroxymethyl-1,3-dioxolan-4-yl)cytosine is the firstexample of an “L”-nucleoside that exhibits anti-tumor activity.(−)-(2S,4S)-1-(2-Hydroxymethyl-1,3-dioxolan-4-yl)cytosine has thestructure illustrated in Formula I.

It has been discovered that(−)-(2S,4S)-1-(2-hydroxymethyl-1,3-dioxolan-4-yl)cytosine exhibitssignificant activity against cancer cells and exhibits low toxicitytoward healthy cells in the host. Nonlimiting examples of cancers thatcan be treated with this compound include lung, colorectal, breast,prostate, bladder, pancreas, ovarian, leukemia, and lymphoma.

In an alternative embodiment, a method and composition for the treatmentof cancer in humans and other host animals is disclosed that includesadministering an effective amount of a compound of the formula:

wherein R is F, Cl, —CH₃, —C(H)═CH₂, —C═CH, or —C═N and R¹ is hydrogen,alkyl, acyl, monophosphate, diphosphate, or triphosphate, or apharmaceutically acceptable derivative thereof, optionally in apharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 indicates the ID₅₀ of (−)-OddC and a combination of (−)-OddC+THU(tetrahydrouridine, a cytidine deaminase inhibitor) on colon cancercells. The graph plots growth inhibition as a percentage of controlgrowth vs. concentration (μM). In the graph, the data for (−)-OddC aloneis represented by () and the data for (−)-OddC+THU is represented by(−−▴−−).

FIG. 2 is a graph of tumor growth weight for mouse carcinoma (Colon 38)treated twice a day with (−)-OddC in a dosage amount of 25 mg/kgbid. Thegraph plots tumor growth as a percentage of original tumor weight vs.days. Treatment of the mice occurred in days 1, 2, 3, 4 and 5. In thegraph, the data for the control (no administration of (−)-OddC) isrepresented by (), the data for (−)-OddC is represented by (−−▴−−).

FIG. 3 indicates the survival rate of P388 leukemic mice that have beentreated with (−)-OddC. The graph plots percentage of survival vs. daystreated. Treatment of the mice occurred in days 1, 2, 3, 4 and 5. In thegraph, the survival rate of the control (no administration of (−)-OddC)is represented by (), the survival rate of those administered (−)-OddCat 25 mg/kgbid twice a day is represented by (−−▴−−), and the survivalrate of mice administered (−)-OddC once a day at 50 mg/kgbid isrepresented by (◯).

FIG. 4 is a plot of the relative sensitivity of certain cancer celllines to (−)-OddC on the basis of GI50. Bars extending to the rightrepresent sensitivity of the cell line to (−)-OddC in excess of theaverage sensitivity of all tested cell lines. Since the bar scale islogarithmic, a bar 2 units to the right implies the compound achievedGI50 for the cell line at a concentration one-hundredth the meanconcentration required over all cell lines, and thus the cell line isunusually sensitive to (−)-OddC. Bars extending to the leftcorrespondingly imply sensitivity less than the mean.

DETAILED DESCRIPTION OF THE INVENTION

The invention as disclosed herein is a method and composition for thetreatment of tumors, and in particular, cancer in humans or other hostanimals, that includes administering an effective amount of(−)-(2S,4S)-1-(2-hydroxymethyl-1,3-dioxolan-4-yl)cytosine, aphysiologically acceptable derivative of the compound, including a 5′ orN⁴ alkylated or acylated derivative, or a physiologically acceptablesalt thereof, optionally in a pharmaceutically acceptable carrier.

(−)-(2S,4S)-1-(2-Hydroxymethyl-1,3-dioxolan-4-yl)cytosine is referred toas an “L”-nucleoside. Since the 2 and 5 carbons of the dioxolane ringare chiral, their nonhydrogen substituents (CH₂OH and the cytosine base,respectively) can be either cis (on the same side) or trans (on oppositesides) with respect to the dioxolane ring system. The four opticalisomers therefore are represented by the following configurations (whenorienting the dioxolane moiety in a horizontal plane such the oxygen inthe 3-position is in front): cis (with both groups “up”, whichcorresponds to the configuration of naturally occurring nucleosides,referred to as a “D”-nucleoside), cis (with both groups “down”, which isthe non-naturally occurring configuration, referred to as an“L”-nucleoside), trans (with the C2 substituent “up” and the C5substituent “down”), and trans (with the C2 substituent “down” and theC5 substituent “up”). It is believed that(−)-(2S,4S)-1-(2-hydroxymethyl-1,3-dioxolan-4-yl)cytosine or itsderivative is the first example of an “L”-nucleoside that exhibitsanti-tumor activity. This is surprising, in light of the fact that this“L” nucleoside configuration does not occur in nature.

As used herein, the term “enantiomerically enriched” refers to anucleoside composition that includes at least approximately 95%, andpreferably approximately 97%, 98%, 99%, or 100% of a single enantiomerof that nucleoside. In a preferred embodiment,(−)-(2S,4S)-1-(2-hydroxymethyl-1,3-dioxolan-4-yl)cytosine is provided asthe indicated enantiomer and substantially in the absence of itscorresponding enantiomer (i.e., in enantiomerically enriched, includingenantiomerically pure form).

The active compound can be administered as any derivative that uponadministration to the recipient, is capable of providing directly orindirectly, the parent (−)-OddC compound, or that exhibits activityitself. Nonlimiting examples are the pharmaceutically acceptable salts(alternatively referred to as “physiologically acceptable salts”) of(−)-OddC, the 5-derivatives as illustrated above, and the 5′ and N⁴acylated or alkylated derivatives of the active compound (alternativelyreferred to as “physiologically active derivatives”). In one embodiment,the acyl group is a carboxylic acid ester (—C(O)R) in which thenon-carbonyl moiety of the ester group is selected from straight,branched, or cyclic alkyl (typically C₁ to C₁₈, and more typically C₁ toC₅) alkaryl, aralkyl, alkoxyalkyl including methoxymethyl, aralkylincluding benzyl, aryloxyalkyl such as phenoxymethyl; aryl includingphenyl optionally substituted with halogen, C₁ to C₄ alkyl or C₁ to C₄alkoxy; sulfonate esters such as alkyl or aralkyl sulphonyl includingmethanesulfonyl, the mono, di or triphosphate ester, trityl ormonomethoxytrityl, substituted benzyl, trialkylsilyl (e.g.dimethyl-t-butylsilyl) or diphenylmethylsilyl. Aryl groups in the estersoptimally comprise a phenyl group.

Specific examples of pharmaceutically acceptable derivatives of(−)-O-ddC include, but are not limited to:

wherein R is F, Cl, —CH₃, —C(H)═CH₂, —C═CH, or —C═N, and R₁ and R₂ areindependently selected from the group consisting of hydrogen, alkyl andacyl, specifically including but not limited to methyl, ethyl, propyl,butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, t-butyl,isopentyl, amyl, t-pentyl, 3-methylbutyryl, hydrogen succinate,3-chlorobenzoate, cyclopentyl, cyclohexyl, benzoyl, acetyl, pivaloyl,mesylate, propionyl, butyryl, valeryl, caproic, caprylic, capric,lauric, myristic, palmitic, stearic, oleic, and amino acids includingbut not limited to alanyl, valinyl, leucinyl, isoleucinyl, prolinyl,phenylalaninyl, tryptophanyl, methioninyl, glycinyl, serinyl,threoninyl, cysteinyl, tyrosinyl, asparaginyl, glutaminyl, aspartoyl,glutaoyl, lysinyl, argininyl, and histidinyl. In a preferred embodiment,the derivative is provided as the indicated enantiomer and substantiallyin the absence of its corresponding enantiomer (i.e., inenantiomerically enriched, including enantiomerically pure form).

(−)-OddC or its derivative can be provided in the form ofpharmaceutically acceptable salts. As used herein, the termpharmaceutically acceptable salts or complexes refers to salts orcomplexes of (−)-OddC or its derivatives that retain the desiredbiological activity of the parent compound and exhibit minimal, if any,undesired toxicological effects. Nonlimiting examples of such salts are(a) acid addition salts formed with inorganic acids (for example,hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid,nitric acid, and the like), and salts formed with organic acids such asacetic acid, oxalic acid, tartaric acid, succinic acid, malic acid,ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid,polyglutamic acid, naphthalenesulfonic acids, naphthalenedisulfonicacids, and polygalacturonic acid; (b) base addition salts formed withpolyvalent metal cations such as zinc, calcium, bismuth, barium,magnesium, aluminum, copper, cobalt, nickel, cadmium, sodium, potassium,and the like, or with an organic cation formed fromN,N-dibenzylethylene-diamine, ammonium, or ethylenediamine; or (c)combinations of (a) and (b); e.g., a zinc tannate salt or the like.

Modifications of the active compound, specifically at the N⁴ and 5′-0positions, can affect the solubility, bioavailability and rate ofmetabolism of the active species, thus providing control over thedelivery of the active species. Further, the modifications can affectthe anticancer activity of the compound, in some cases increasing theactivity over the parent compound. This can easily be assessed bypreparing the derivative and testing its anticancer activity accordingto the methods described herein, or other method known to those skilledin the art.

II. Preparation of the Active Compounds

(−)-OddC can be prepared according to the method disclosed in detail inPCT International Publication No. WO 92/18517, published on Oct. 29,1992, or by the method disclosed in Scheme 1 and working examples 1-7provided below, or by any other method known to those skilled in theart. These methods, or other known methods, can be adapted for thepreparation of the exemplified derivatives of (−)-OddC.

EXAMPLE 1 Preparation of 6-Anhydro-L-gulose

6-Anhydro-L-gulose was prepared in one step from L-gulose by thetreatment of L-gulose with an acid, e.g., 0.5N HCl, in 60% yield (Evans,M. E., et al., Carbohydr. Res. (1973), 28, 359). Without selectiveprotection, as was done before (Jeong, L. S. et al. Tetrahedron Lett.(1992), 33, 595 and Beach, J. W. et al. J. Org. Chem. (1992, in press),(2) was directly converted to dioxolane triol (3) by oxidation withNaIO₄, followed by reduction with NaBH₄, which without isolation, wasconverted to isopropylidene derivative (4). Benzoylation to (5),deprotection to (6), and oxidation of diol (6) gave the acid (7).Oxidative decarboxylation of (7) with Pb(OAc)₄ in dry THF gave theacetate (8), the key intermediate in good yield. The acetate wascondensed with the desired pyrimidines (e.g., silylated thymine andN-acetylcytosine) in the presence of TMSOTf to afford an α,β-mixture,which was separated on a silica gel column to obtain the individualisomers (9 and 10). Debenzoylation with methanolic ammonia gave thedesired (−)-OddC (11).

EXAMPLE 2 Preparation of (−)-1,6-Anhydro-α-L-gulopyranose (2)

A mixture of L-gulose (1) (33 g, 0.127 mol) and 0.5 N HCl (330 mL, 0.165mol) was refluxed for 20 hours. The mixture was cooled and neutralizedto pH 6 by a resin (Dowex-2, HCO₃-form) with air bubbling. The resin wasrecycled by washing with 10% HCl, water, methanol, water and saturatedNaHCO₃ solution. The reaction mixture was filtered and the resin waswashed with water (500 mL). The combined filtrate was concentrated todryness and dried in vacuo overnight. The residue was purified over acolumn (5 cm depth, silica gel, mesh, CHCl₃-CH₃OH, 10:1) to give aslightly yellow solid, which was recrystallized from absolute alcohol togive a colorless solid (2) [R_(f)=0.43 (CHCl₃-CH₃OH, 5:1), 7.3 g,35.52%]. The L-gulose R_(f)=0.07, 11 g) obtained was recycled to give(2) (5 g, total yield 60%):mp 142.5-145° C.; ¹H NMR (DMSO-d₆) δ3.22-3.68 (m, 4H, H-2, -3, -4 and -6a) , 3.83 (d, J_(6b,6a)=7.25 Hz, 1H,H_(b)-6) 4.22 (pseudo t, J_(5,6a)=4.61 and 4.18 Hz, H, H-5), 4.46 (d,J_(2-OH,2)=6.59 Hz, 1H, 2-OH, exchangeable with D₂O), 4.62 (d,J_(3-OH,3)=5.28 Hz, 1H, 3-OH, exchangeable with D₂O), 5.07 (d,J_(4-OH4)=4.84 Hz, 1H, 4-OH, exchangeable with D₂O), 5.20 (d,J_(1,2)=2.19 Hz, 1H, H-1). [α]_(D) ²⁵-50.011 (c, 1.61, CH₃OH).

EXAMPLE 3 Preparation of(−)-(1′S,2S,4S)-4-(1,2-Dihydroxyethyl-1,2-O-Isopropylidene)-2-hydroxymethyl)-dioxolane(4)

A solution of NaIO₄ (22.36 g, 0.1 mol) in water (300 mL) was added in adropwise manner over 10 minutes to a solution of (2) (11.3 g, 0.07 mol)in methanol (350 mL) cooled to 0° C. The mixture was stirredmechanically for 15 minutes. NaBH₄ (7.91 g, 0.21 mol) was added to thismixture and the reaction mixture was stirred for 10 minutes at 0° C. Thewhite solid was filtered off and the solid was washed with methanol (300mL). The combined filtrate was neutralized by 0.5 N HCl (−200 mL) andconcentrated to dryness. The residue was dried in vacuo overnight. Thesyrupy residue was triturated with methanol-acetone (1:5 , 1200 mL)using a mechanical stirrer (5 hours) and the white solid (1st.) wasfiltered off. The filtrate was concentrated to dryness and the residuewas dissolved in acetone (500 mL) and followed by p-toluene sulfonicacid (6.63 g, 0.035 mol). After stirring for 6 hours, the mixture wasneutralized by triethylamine, the solid (2nd.) was filtered off and thefiltrate was concentrated to dryness. The residue was dissolved in ethylacetate (350 mL) and washed with water (50 mL×2) , dried (MgSO₄),filtered, and evaporated to give crude (4) (3.6 g) as a yellowish syrup.The water layer was concentrated to dryness and dried in vacuo. Thesolid obtained (1st and 2nd) was combined with the dried water layer andrecycled by stirring for 1 hour in 10% methanol-acetone (900 mL) andp-toluene sulfonic acid (16 g, 0.084 mol) to yield crude (4) (5.6 g).The crude (4) was purified by a dry column over silica gel (CH₃OH-CHCl₃,1%-5%) to give (4) [R_(f)=0.82 (CHCl₃-CH₃OH, 10:1), 8.8 g, 61.84%] as acolorless oil. ¹H NMR(DMSO-d₆) δ 1.26 and 1.32 (2×s, 2×3 H,isopropylidene), 3.41 (dd, J_(CH2OH,OH)=6.04 Hz, J_(CH2OH,2)=3.96 Hz,2H, CH₂OH), 3.56-4.16 (m, 6H, H-4, -5, -1′ and -2′), 4.82 (t,J_(OH,CH2)=6.0 Hz, 1 H, CH₂OH, exchangeable with D₂O), 4.85 (t,J_(2OH,CH2OH)=3.96 Hz, 1H, H-2). [α]_(D) ²⁵-12.48 (c, 1.11, CHCl₃),Anal, Calcd for C₉H₁₆O₅:C, 52.93; H, 7.90. Found: C, 52.95; H, 7.86.

EXAMPLE 4 Preparation of(+)-(1′S,2S,4S)-4-(1,2-Dihydroxymethyl-1,2-O-Isopropylidene)-2-(O-benzoyloxymethyl)-dioxolane(5)

Benzoyl chloride (6.5 mL, 0.056 mol) was added in a dropwise manner to asolution of (4) (8.5 g, 0.042 mol) in pyridine-CH₂Cl₂ (1:2, 120 mL) at0° C. and the temperature was raised to room temperature. After stirringfor 2 hours, the reaction was quenched with methanol (10 mL) and themixture was concentrated to dryness in vacuo. The residue was dissolvedin CH₂Cl₂ (300 mL) and washed with water (100 mL×2), brine, dried(MgSO₄), filtered, evaporated to give a yellowish syrup, which waspurified by silica gel column chromatography (EtOAc-Hexane 4% -30%) toyield (5) [R_(f)=0.45 (Hexane-EtOAc, 3:1), 10.7 g, 83.4%] as a colorlessoil. 1H NMR (CDCl₃) δ 1.35 and 1.44 (2×s, 2×3H, isopropylidene) 3.3-4.35(m 6H, H-4, -5, -1′ and -2′), 4.44 (d, J=3.96 Hz, 2H, CH₂-OBz), 5.29 (t,J=3.74 Hz, 1H, H-2), 7.3-7.64, 8.02-8.18 (m, 3H, 2H, -OBz). [α]_(D)²⁵+10.73 (c, 1.75, CH₃OH). Anal. Calcd for C₁₆H₂₀O₆:C, 62.33; H, 6.54.Found: C, 62.39; H, 6.54.

EXAMPLE 5 Preparation of(+)-(1′S,2S,4S)-4-(1,2-Dihydroxyethyl)-2-(O-benzoyloxymethyl)-dioxolane(6)

A mixture of (5) (5.7 g. 0.018 mol) and p-toluene sulfonic acid (1.05 g.0.0055 mol) in methanol (70 mL) was stirred at room temperature for 2hours. The reaction was not completed, so the solvent was evaporated tohalf of the original volume and additional methanol (50 mL) andp-toluene sulfonic acid (0.7 g, 3.68 mmol) were added. After stirringfor one more hour, the reaction mixture was neutralized with triethylamine and the solvent was evaporated to dryness.

The residue was purified by silica gel column chromatography(Hexane-EtOAC, 10%-33%) to give (6) [R_(f)=0.15 (Hexane-EtOAc, 1:1),4.92 g, 99.2%] as a colorless syrup ¹H NMR (DMSO-d₆) )δ 3.43 (m, 2H,H-2′), 3.67-4.1 (m, 4H, H-4, -5 and -1′), 4.32 (d, J=3.73 Hz, 2H,CH₂-OBz), 4.60 (t, J=5.72 Hz, 2′-OH, exchangeable with D₂O), 5.23 (t,J=3.96 Hz, 1H, H-2), 7.45-7.7, 7.93-8.04 (m, 3H, 2H, -OBz), [α]_(D)²⁵+9.16 (c, 1.01, CHCl₃). Anal. Calcd for C₁₃H₁₆O₆:C, 58.20; H, 6.01.Found: c,58.02; H.6.04.

EXAMPLE 6 Preparation of (−)-(2S,4S) and (2S,4R)-4-Acetoxy-2-(O-benzoyloxymethyl)-dioxolane (8)

A solution of NaIO₄ (10.18 g, 0.048 mol) in water (120 mL) was added toa solution of (6) (3.04 g, 0.011 mol) in CCl₄:CH₃CN (1:1, 160 mL),followed by RuO₂ hydrate (0.02 g). After the reaction mixture wasstirred for 5 hours, the solid was removed by filtration over Celite andthe filtrate was evaporated to ⅓ volume. The residue was dissolved inCH₂Cl₂ (100 mL) and the water layer was extracted with CH₂Cl₂ (100mL×2). The combined organic layer was washed with brine (50 mL), dried(MgSO₄), filtered, evaporated to dryness and dried in vacuo for 16 hoursto give crude (7) (2.6 g, 91%)

To a solution of crude (7) (2.6, 0.01 mol) in dry THF (60 mL) were addedPb(OAc)₄(5.48 g, 0.0124 mol) and pyridine (0.83 mL, 0.0103 mol) under N₂atmosphere. The mixture was stirred for 45 minutes under N₂ and thesolid was removed by filtration. The solid was washed with ethyl acetate(60 mL) and the combined organic layer was evaporated to dryness. Theresidue was purified by silica gel column chromatography (Hexane-EtOAc,2:1) to yield (8) [R_(f)=0.73 and 0.79 (Hexane-EtOAc, 2:1), 1.9 g,69.34%] as a colorless oil. ¹H NMR (CDCl₃) δ 1.998, 2.11 (2×s, 3H,-OAc), 3.93-4.33 (m, 2H, H-5), 4.43, 4.48 (2×d, J=3.73, 3.74 Hz, 2H,CH₂OBz) 5.46, 5.55 (2×t, J=4.18, 3.63 Hz, 1H, H-2), 6.42 (m, 1H, H-4),7.33-759, 8.00-8.15 (m, 3H, 2H, -OBz). [α]_(D) ²⁵-12.53 (c, 1.11,CHCl₃). Anal. Calcd for C₁₃H₁₄O₆; C, 58.64; H, 5.30. Found C, 58.78; H,5.34.

EXAMPLE 7 Preparation of(−)-(2S,4S)-1-[2-(benzoyl)-1,3-dioxolan-4-yl]cytosine (9) and(+)-(2S,4R)-1-[2-(benzyloxy)-1,3-dioxolan-4-yl)cytosine (10)

A mixture of N⁴-acetylcytosine (1.24 g, 7.52 mmol) in dry dichloroethane(20 mL), hexamethyldisilazane (15 mL), and ammonium sulfate (cat.amount) was refluxed for 4 hours under a nitrogen atmosphere. Theresulting clear solution was cooled to room temperature. To thissilylated acetylcytosine was added a solution of (8) (1.0 g, 3.76 mmol)in dry dichloroethane (10 mL) and TMSOTf (1.46 mL 7.55 mmol). Themixture was stirred for 6 hours. Saturated NaHCO₃ (10 mL) was added andthe mixture was stirred for another 15 minutes and filtered through aCelite pad. The filtrate was evaporated and the solid was dissolved inEtOAc and washed with water and brine, dried, filtered and evaporated togive the crude product. This crude product was purified on a silicacolumn (5% CH₃OH/CHCl₃) to yield a pure α,β mixture of (9) and (10)(0.40 g, 30%) and the α,β mixture of (13) and (14) (0.48 g, 40%). Themixture of (14) was reacetylated for separation, the combined α,βmixture was separated by a long silica column (3% CH₃OH/CHCl₃) to yield(9) (0.414 g, 30.7%) and (10) (0.481 g, 35.6%) as foams. These foamswere triturated with CH₃OH to obtain white solids. 9: UV (CH₃OH) λ max298 nm; Anal. (C₁₇H₁₇N₃O₈) C, H, N. 10: UV (CH₃OH) λ max 298 nm.

EXAMPLE 8 Preparation of(−)-(2S,4S)-1-(2-Hydroxymethyl-1,3-dioxolan-4-yl)cytosine (11)

A solution of (9) (0.29 g, 0.827) in CH₃OH/NH₃ (50 mL, saturated at 0°C.) was stirred at room temperature for 10 hours. The solvent wasevaporated and the crude (11) was purified on preparative silica plates(20% CH₃OH/CHCl₃) to give an oil. This was crystallized fromCH₂Cl₂/hexane to give (11) (0.136 g, 77.7%) as a white solid. UV λ max278.0 nm (ε 11967) (pH 2), 270.0 nm (ε 774) (pH 7), 269.0 nm (ε 8379)(pH 11) ; Anal. (C₈H₁₁N₃O₄)C,H,N.

II. Pharmaceutical Compositions

Humans, equines, canines, bovines and other animals, and in particular,mammals, suffering from cancer can be treated by administering to thepatient an effective amount of (−)-OddC or its derivative or apharmaceutically acceptable salt thereof optionally in apharmaceutically acceptable carrier or diluent, either alone, or incombination with other known anticancer or pharmaceutical agents. Thistreatment can also be administered in conjunction with otherconventional cancer therapies, such as radiation treatment or surgery.

These compounds can be administered by any appropriate route, forexample, orally, parenterally, intravenously, intradermally,subcutaneously, or topically, in liquid, cream, gel, or solid form, orby aerosol form.

The active compound is included in the pharmaceutically acceptablecarrier or diluent in an amount sufficient to deliver to a patient atherapeutically effective amount for the desired indication, withoutcausing serious toxic effects in the patient treated. A preferred doseof the compound for all of the herein-mentioned conditions is in therange from about 10 ng/kg to 300 mg/kg, preferably 0.1 to 100 mg/kg perday, more generally 0.5 to about 25 mg per kilogram body weight of therecipient per day. A typical topical dosage will range from 0.01-3%wt/wt in a suitable carrier.

The compound is conveniently administered in any suitable unit dosageform, including but not limited to one containing 1 to 3000 mg,preferably 5 to 500 mg of active ingredient per unit dosage form. A oraldosage of 25-250 mg is usually convenient.

The active ingredient is preferably administered to achieve peak plasmaconcentrations of the active compound of about 0.00001-30 mM, preferablyabout 0.1-30 μM. This may be achieved, for example, by the intravenousinjection of a solution or formulation of the active ingredient,optionally in saline, or an aqueous medium or administered as a bolus ofthe active ingredient.

The concentration of active compound in the drug composition will dependon absorption, distribution, inactivation, and excretion rates of thedrug as well as other factors known to those of skill in the art. It isto be noted that dosage values will also vary with the severity of thecondition to be alleviated. It is to be further understood that for anyparticular subject, specific dosage regimens should be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of thecompositions, and that the concentration ranges set forth herein areexemplary only and are not intended to limit the scope or practice ofthe claimed composition. The active ingredient may be administered atonce, or may be divided into a number of smaller doses to beadministered at varying intervals of time.

Oral compositions will generally include an inert diluent or an ediblecarrier. They may be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound or its prodrug derivative can be incorporated with excipientsand used in the form of tablets, troches, or capsules. Pharmaceuticallycompatible binding agents, and/or adjuvant materials can be included aspart of the composition.

The tablets, pills, capsules, troches and the like can contain any ofthe following ingredients, or compounds of a similar nature: a bindersuch as microcrystalline cellulose, gum tragacanth or gelatin; anexcipient such as starch or lactose, a dispersing agent such as alginicacid, Primogel, or corn starch; a lubricant such as magnesium stearateor Sterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring. When the dosage unitform is a capsule, it can contain, in addition to material of the abovetype, a liquid carrier such as a fatty oil. In addition, dosage unitforms can contain various other materials which modify the physical formof the dosage unit, for example, coatings of sugar, shellac, or entericagents.

The active compound or pharmaceutically acceptable salt thereof can beadministered as a component of an elixir, suspension, syrup, wafer,chewing gum or the like. A syrup may contain, in addition to the activecompounds, sucrose as a sweetening agent and certain preservatives, dyesand colorings and flavors.

The active compound or pharmaceutically acceptable salts thereof canalso be mixed with other active materials that do not impair the desiredaction, or with materials that supplement the desired action, such asother anticancer agents, antibiotics, antifungals, antiinflammatories,or antiviral compounds.

Solutions or suspensions used for parenteral, intradermal, subcutaneous,or topical application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. The parental preparationcan be enclosed in ampoules, disposable syringes or multiple dose vialsmade of glass or plastic.

If administered intravenously, preferred carriers are physiologicalsaline or phosphate buffered saline (PBS).

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art.

Liposomal suspensions may also be pharmaceutically acceptable carriers.These may be prepared according to methods known to those skilled in theart, for example, as described in U.S. Pat. No. 4,522,811 (which isincorporated herein by reference in its entirety). For example, liposomeformulations may be prepared by dissolving appropriate lipid(s) (such asstearoyl phosphatidyl ethanolamine, stearoyl phosphatidyl choline,arachadoyl phosphatidyl choline, and cholesterol) in an inorganicsolvent that is then evaporated, leaving behind a thin film of driedlipid on the surface of the container. An aqueous solution of the activecompound are then introduced into the container. The container is thenswirled by hand to free lipid material from the sides of the containerand to disperse lipid aggregates, thereby forming the liposomalsuspension.

III. Biological Activity

A wide variety of biological assays have been used and are accepted bythose skilled in the art to assess anti-cancer activity of compounds.Any of these methods can be used to evaluate the activity of thecompounds disclosed herein.

One common method of assessing activity is through the use of theNational Cancer Institute's (“NCI”) test panels of cancer cell lines.These tests evaluate the-in vitro anti-cancer activity of particularcompounds, and provide predictive data with respect to the use of testedcompounds in vivo. Other assays include in vivo evaluations of thecompound's effect on human or mouse tumor cells implanted into orgrafted onto nude mice.

A. In Vivo Activity of (−)-OddC

(−)-OddC was tested for anticancer activity in vivo against the P388leukemia cell line and the C38 colon cancer cell line. Examples 8 and 9provide the experimental details and results of these tests.

EXAMPLE 9 In Vivo Treatment Of Leukemia P388 Cells with (−)-O-ddC

10 ⁶ Leukemia P388 cells were implanted ip to BDF1 mice obtained fromSouthern Research Institute, Alabama. (−)-OddC was administered ip twicedaily for five days starting one day after tumor cell implantation.Using this protocol, 75 mg/kg/dose was shown to be toxic to the mice.

FIG. 3 and Table 1 show the results of these studies. In FIG. 3, ()represents the data for the control (untreated animals), (−−▴−−)represents the survival rate of those administered (−)-OddC at 25mg/kgbid twice a day, and (◯) represents the survival rate of miceadministered (−)-OddC once a day at 50 mg/kgbid. Of the six mice treatedwith 25 mg/kg/dose of (−)-OddC, there is one long term survivor, and thelife span of the remaining five mice was increased by 103%.

TABLE 1 Mean Dosage^(a) Survival ILS^(b) Death Cures^(c)/ Group (mg/kg)Route Time (days) (%) Time (day) Total Control — — 13.3 — 11, 12, 13 0/613, 13, 18 -OddC 25 × 2 × 5 ip 27 103 18, 20, 22, 1/6 25, 33, 45Inoculum: 10⁶ P388 cells were inoculated into each mouse ip on day 0^(a)Treatment was given twice a day on days 1 to 5 ^(b)Increased LifeSpan percent above control ^(c)Survivors equal or greater than 45 daylife span

EXAMPLE 10 In Vivo Treatment Of Colon 38 Tumor Cells with (−)-OddC

Colon 38 tumor cells were implanted sc to BDF1 mice. (−)-OddC wasadministered to the mice twice daily for five days, at a dosage of 25mg/kg/dose. The colon tumor cell growth was retarded as shown in FIG. 2.In FIG. 2, () represents the data from the control animals, and (▴)represents the data from the mice treated with (−)-OddC.

B. In Vitro Testing of (−)-OddC

(−)-OddC was evaluated in the NCI's cancer screening program. The testmeasures the inhibition of various cancer cell lines at variousconcentrations of (−)-OddC. The cell lines which were tested are setforth in Table 2.

Table 2 also provides the concentration at which GI50 and TGI wereobserved in the tested cell lines. GI50, TGI and LC50 are valuesrepresenting the concentrations at which the PG (percent of growthinhibition), defined below, is +50, 0, and −50, respectively. Thesevalues were determined by interpolation from dose response curvesestablished for each cell line, plotted as a function of PG v. log₁₀concentration of (−)-OddC.

PG is the measured effect of (−)-OddC on a cell line and was calculatedaccording to one of the following two expressions:

-   -   If (Mean OD_(test)-Mean OD_(tzero))≧0. then    -   PG=100×(Mean OD_(test)-Mean OD_(tzero))/(Mean OD_(ctrl)-Mean        OD_(tzero))    -   If (Mean OD_(test)-Mean OD_(tzero))<0. then    -   PG=100×(Mean OD_(text)-Mean OD_(tzero))/(Mean OD_(tzero))

Where:

-   -   Mean OD_(tzero)=The average of optical density measurements of        SRB-derived color just before exposure of cells to the test        compound.    -   Mean OD_(text)=The average of optical density measurements of        SRB-derived color after 48 hours exposure of cells to the test        compound.    -   Mean OD_(ctrl)=The average of optical density measurements of        SRB-derived color after 48 hours with no exposure of cells to        the test compound.

In Table 2, the first two columns describe the subpanel (e.g., leukemia)and cell line (e.g., CCRF-CEM) which were treated with (−)-OddC. Column3 indicates the log₁₀ at which GI50 occurred and column 4 indicates thelog₁₀ at which TGI occurred. If these response parameters could not beobtained by interpolation, the value given for each response parameteris the highest concentration tested and is preceded by a “>” sign. Forexample, if all the PG at al concentrations of (−)-OddC given to aparticular cell line exceeds +50, then this parameter can not beobtained by interpolation.

TABLE 2 Panel Cell Line Log₁₀GI50 Log₁₀TGI Leukemia CCRF-CEM−6.64 >−4.00 RL-60(TB) −6.28 >−4.00 K-562 −4.59 >−4.00 BSOLT-4 −6.66−4.39 RPMI-2.26 −4.03 >−4.00 SR −5.95 >−4.00 Non-Small A549/ATCC−6.01 >−4.00 Cell Lung Cancer BKVX >−4.00 >−4.00 HOP-62 −6.23 −4.71NCI-H23 −4.92 >−4.00 NCI-H322M >−4.00 >−4.00 NCI-H460 −4.32 >−4.00NCI-H522 −6.06 >−4.00 Colon COLO205 −4.03 >−4.00 Cancer HCT-116−5.23 >−4.00 HCT-15 −5.39 >−4.00 HT29 >−4.00 >−4.00 K2112 >−4.00 >−4.00CNS Cancer SP-268 −5.18 >−4.00 SP-295 −6.24 >−4.00 SNB-19 −5.71 >−4.00U251 −4.91 >−4.00 Melanoma LOX D6VI −6.39 >−4.00 MALME-3M −4.51 >−4.00M14 −6.27 −5.07 SK-MEL-28 −4.31 >−4.00 SK-MEL-5 −4.91 >−4.00UACC-257 >−4.00 >−4.00 UACC-62 −5.53 >−4.00 Ovarian OROV1 −4.03 >−4.00Cancer OVCAR-3 −4.44 >−4.00 OVCAR-4 >−4.00 >−4.00 OVCAR-5 −4.41 >−4.00OVCAR-8 −5.82 >−4.00 SK-OV-3 −5.35 >−4.00 Renal 785-4 −5.36 >−4.00Cancer ACHN −6.46 >−4.00 CAKI-1 −6.65 −4.87 RXF-393 −6.17 >−4.00 SN12C−6.27 >−4.00 TK-30 >−4.00 >−4.00 UO-31 −5.60 >−4.00 Prostate PC-3−6.29 >−4.00 Cancer DU-145 −6.97 >−4.00 Breast MCF7 −5.95 >−4.00 CancerMCF7/ADR-RES −4.97 >−4.00 MDA-MB- >−4.00 >−4.00 231/ATCCHS578T >−4.00 >−4.00 MDA-MB-435 −4.62 >−4.00 MDA-N −4.33 >−4.00 BT-549−4.59 >−4.00 T-47D >−4.00 >−4.00

FIG. 4 is a graph that displays the relative selectivity of (−)-OddC fora particular cell line. Bars extending to the right representsensitivity of the cell line to (−)-OddC in excess of the averagesensitivity of all tested cell lines. Since the bar scale islogarithmic, a bar 2 units to the right implies the compound exhibited aGI50 for the cell line at a concentration one-hundredth the meanconcentration required over all cell lines, and thus the cell line isunusually sensitive to (−)-OddC. Bars extending to the leftcorrespondingly imply sensitivity less than the mean. These cell linescan be easily determined from Table 2, as the log₁₀ concentration willbe preceded by a “>”.

It can be seen from FIG. 4 that at least one cell line of each type ofcancer cell tested exhibited sensitivity to (−)-OddC. Certain prostatecancer cell lines, leukemia cell lines, and colon cell lines showextreme sensitivity to (−)-OddC.

EXAMPLE 11 Comparison of (−)-OddC and AraC

As discussed in the Background of the Invention, cytosine arabinoside(also referred to as Cytarabin, araC, and Cytosar) is a nucleosideanalog of deoxycytidine used in the treatment of acute myeloid leukemia.It is also active against acute lymphocytic leukemia, and to a lesserextent, is useful in chronic myelocytic leukemia and non-Hodgkin'slymphoma. The primary action of araC is inhibition of nuclear DNAsynthesis. It was of interest to compare the toxicity to tumor cells of(−)-OddC and AraC.

Cells in logarithmic growth were plated at a density of 5000cells/mL/well in 24-well plates. Drugs were added to the cells atdifferent dosages and cultures were maintained for a period of threegenerations. At the end of this time, methylene blue assays wereperformed and/or cell numbers were directly counted. Methylene blue is adie which binds in a stoichiometric manner to proteins of viable cellsand can be used to indirectly quantitate cell number (Finlay, 1984).IC₅₀ values were determined by interpolation of the plotted data. Eachvalue shown is the mean±standard deviation of five experiments with eachdata point done in duplicate.

In all of the tumor cell lines tested, (−)-OddC was more cytotoxic thanAraC. (−)-OddC was significantly more effective than AraC in the KBnasopharyngeal carcinoma cell line and in the two prostate carcinomalines DU-145 and PC-3. HepG2 cells originate from hepatocellularcarcinoma and the 2.2.15 line is derived from HepG2 cells which weretransfected with a copy of the hepatitis B virus genome. CEM cells arederived from acute lymphoblastic leukemia. (−)-OddU, the compound whichwould be formed by the deamination of (−)-OddC was not toxic in any ofthe cell lines tested. Enzymatic studies indicate that, unlike AraCwhose clinical efficacy is greatly diminished by its susceptibility todeamination, (−)-OddC is not a substrate for deaminase.

It has been determined that (−)-OddC can be phosphorylated to mono-, di-and tri-phosphate nucleotide in vivo. It appears that (−)-OddC exhibitsits cellular toxicity in a phosphorylated form because cells that areincapable of phosphorylating the compound are much less sensitive to thecompound. The first enzyme responsible for its phosphorylation is humandeoxycytidine kinase. In vitro enzymatic studies indicate that (−)-OddCcan be phosphorylated by this enzyme.

Unlike araC, (−)-OddC is not deaminated by cytidine deaminase. Thepresence of cytidine deaminase in solid tumor tissues could be a keycontributing factor responsible for the lack of activity of araC insolid tumors. This could partly explain why (−)-OddC is active againstHepG2 cells in nude mice, whereas araC is inactive. It also explains why(−)-OddC has different spectrums of anti-tumor activity from that ofaraC. Furthermore, the presence of cytidine deaminase in thegastointestinal tract could also play an important role in why araCcannot be taken orally. The lack of action of cytidine deaminase to(−)-OddC may explain why (−)-OddC could still have anti-tumor activityif given orally.

BIOCHEMICAL STUDIES OF (-)-OddC In vitro cytotoxicity of AraC, (-)-OddCand (-)-OddU ID₅₀ (μM) Cell Line AraC (-)-OddC (-)-OddU KB 0.152 ± .0100.048 ± .021 >30 DU-145 0.170 ± .035 0.024 ± .020 >30 PC-3 0.200 ± .0780.056 ± .039 >30 HepG2 0.125 ± .013 0.110 ± .050 >30 2.2.15 0.145 ± .0070.110 ± .011 >30 CEM 0.030 ± .010 0.025 ± .030 >30

IV. Use of (−)-OddC in Oligonucleotides and in Antisense Technology

Antisense technology refers in general to the modulation of geneexpression through a process wherein a synthetic oligonucleotide ishybridized to a complementary nucleic acid sequence to inhibittranscription or replication (if the target sequence is DNA), inhibittranslation (if the target sequence is RNA) or to inhibit processing (ifthe target sequence is pre-RNA). A wide variety of cellular activitiescan be modulated using this technique. A simple example is theinhibition of protein biosynthesis by an antisense oligonucleotide boundto mRNA. In another embodiment, a synthetic oligonucleotide ishybridized to a specific gene sequence in double stranded DNA, forming atriple stranded complex (triplex) that inhibits the expression of thatgene sequence. Antisense oligonucleotides can be also used to activategene expression indirectly by suppressing the biosynthesis of a naturalrepressor or directly by reducing termination of transcription.Antisense oligonucleotide Therapy (AOT) can be used to inhibit theexpression of pathogenic genes, including those which are implicated inthe uncontrolled growth of benign or malignant tumor cells or which areinvolved in the replication of viruses, including HIV and HBV.

The stability of the oligonucleotides against nucleases is an importantfactor for in vivo applications. It is known that 3′-exonucleaseactivity is responsible for most of the unmodified antisenseoligonucleotide degradation in serum. Vlassov, V. V., Yakubov, L. A., inProspects for Antisense Nucleic Acid Therapy of Cancers and AIDS, 1991,243-266, Wiley-Liss, Inc., New York; Nucleic Acids Res., 1993, 21, 145.

The replacement of the nucleotide at the 3′-end of the oligonucleotidewith (−)-OddC or its derivative can stabilize the oligonucleotideagainst 3′-exonuclease degradation. Alternatively or in addition, aninternal nucleotide can be replaced by (−)-OddC or its derivative toresist the degradation of the oligonucleotide by endonucleases.

Given the disclosure herein, one of ordinary skill in the art will beable to use (−)-OddC or its derivative to stabilize a wide range ofoligonucleotides against degradation by both exonucleases andendonucleases, including nucleosides used in antisense oligonucleotidetherapy. All of these embodiments are considered to fall within thescope of this invention. Example 11 provides one, non-limiting, exampleof the use of (−)-OddC to resist the activity of a 3′-exonuclease.

EXAMPLE 11 Resistance to 3′-Exonuclease Activity by (−)-OddC

The human cytosolic exonuclease activity from human H9 (T-typelymphocytic leukemic cells) was determined by sequencing gel assay.Briefly, the 3′-terminated substrate was prepared from a 20 or 23base-long DNA primer with the following sequence:

3′-CAATTTTGAATTTCCTTAACTGCC-5′         24                       1The primers were labelled at the 5′-end with

-³²P] ATP, annealed to complementary RNA templates and terminated at the3′ end with dTTP (20 mer) dCTP (23 mer) or (−)-OddCTP (23 mer) in astanding start reaction catalyzed by HIV-1 RT. Under these conditions,the 20 mer was terminated with dTMP (A) the 23 mer was terminated withdCMP (B) or (−)-O-ddCMP(C). These single stranded DNA substrates wereused to assay their susceptibility to the cytoplasmic exonuclease. Theassays were done in 10 μl reactions containing 50 mM Tris-HCl pH 8.0, 1mM MgCl₂, 1 mM dithiothreitol, 0.1 mg/ml bovine serum albumin, 0.18μCi/ml 3′-terminated substrate and 2 μl of the exonuclease (0.03 units).The reactions were incubated at 37° C. for the indicated times andterminated by adding 4 μl 98% formamide, 10 mM EDTA and 0.025%bromophenol blue. The samples were denatured at 100° C. for 5 minutesfollowed by rapid cooling on ice. The unreacted material as well as thereaction products were separated on 15% polyacrylamide/urea sequencinggels and visualized by autoradiography. The oligonucleotide with(−)-OddC at the 3′-end was at least five times more resistant to3′-exonuclease than the other oligonucleotides.

Modifications and variations of the present invention in the treatmentof cancer will be obvious to those skilled in the art from the foregoingdetailed description of the invention. Such modifications and variationsare intended to come within the scope of the appended claims.

1-19. (canceled)
 20. A method for treating a cancer selected from thegroup consisting of lung cancer, colorectal cancer, breast cancer,prostate cancer, bladder cancer, pancreatic cancer, ovarian cancer,uterine cancer, leukemia, cns cancer, melanoma, renal cancer, lymphomaand head and neck cancer in a host animal comprising administering tosaid animal an effective amount of a compound of the formula

wherein R¹ and R² are independently selected from the group consistingof an acyl group in which the non-carbonyl moiety of the ester group isselected from straight, branched, or cyclic C₁ to C₁₈ alkyl,methoxymethyl, phenoxymethyl or phenyl, a sulfonate ester, and mono, diand triphosphate ester said cancer being sensitive to treatment withsaid compound.
 21. The method according to claim 20 wherein said canceris prostate cancer.
 22. The method according to claim 20 wherein saidcancer is lung cancer.
 23. The method according to claim 20 wherein saidcancer is colorectal cancer.
 24. The method according to claim 20wherein said cancer is breast cancer.
 25. The method according to claim20 wherein said cancer is bladder cancer.
 26. The method according toclaim 20 wherein said cancer is pancreatic cancer.
 27. The methodaccording to claim 20 wherein said cancer is ovarian cancer.
 28. Themethod according to claim 20 wherein said cancer is lymphoma.
 29. Themethod according to claim 20 wherein said cancer is leukemia.
 30. Amethod for treating a cancer selected from the group consisting of lungcancer, colorectal cancer, breast cancer, prostate cancer, bladdercancer, pancreatic cancer, ovarian cancer, uterine cancer, leukemia, cnscancer, melanoma, renal cancer, lymphoma and head and neck cancer in ahuman comprising administering to said human an effective amount of acompound of the formula

wherein R¹ and R² are independently selected from the group consistingof an acyl group in which the non-carbonyl moiety of the ester group isa straight, branched, or cyclic C₁ to C₁₈ alkyl group, methoxymethyl,phenoxymethyl or phenyl, a sulfonate ester, and mono, di andtriphosphate ester, said cancer being sensitive to treatment with saidcompound.
 31. The method according to claim 30 wherein said cancer isprostate cancer.
 32. The method according to claim 30 wherein saidcancer is lung cancer.
 33. The method according to claim 30 wherein saidcancer is colorectal cancer.
 34. The method according to claim 30wherein said cancer is breast cancer.
 35. The method according to claim30 wherein said cancer is bladder cancer.
 36. The method according toclaim 30 wherein said cancer is pancreatic cancer.
 37. The methodaccording to claim 30 wherein said cancer is ovarian cancer.
 38. Themethod according to claim 30 wherein said cancer is lymphoma.
 39. Themethod according to claim 30 wherein said leukemia is acute myeloidleukemia.